Bridging the gap between in silico and cell-based analysis of the nuclear factor-jB signaling pathway by in vitro studies of IKK2 Adaoha E C Ihekwaba1,4, Stephen J Wilkinson1, Dominic Waithe3, David S Broomhead2, Peter Li1, Rachel L Grimley3 and Neil Benson3 School of Chemistry, The University of Manchester, Faraday Building, UK School of Mathematics, The University of Manchester, UK Pfizer Global Research and Development, Sandwich, UK VBI, Virginia Tech, Blacksburg, VA, USA Keywords enzyme kinetics; in silico; in vitro; nuclear factor-jB regulation; signal transduction Correspondence A E C Ihekwaba, VBI, Virginia Tech, Washington Street, Blacksburg, VA, USA Fax: +1 540 2312606 Tel: +1 540 2310795 E-mail: ihekwaba@vbi.vt.edu (Received 13 July 2006, revised 19 December 2006, accepted 22 January 2007) doi:10.1111/j.1742-4658.2007.05713.x Previously, we have shown by sensitivity analysis, that the oscillatory behavior of nuclear factor (NF-jB) is coupled to free IkappaB kinase-2 (IKK2) and IkappaBalpha(IjBa), and that the phosphorylation of IjBa by IKK influences the amplitude of NF-jB oscillations We have performed further analyses of the behavior of NF-jB and its signal transduction network to understand the dynamics of this system A time lapse study of NF-jB translocation in 10 000 cells showed discernible oscillations in levels of nuclear NF-jB amongst cells when stimulated with interleukin (IL-1a), which suggests a small degree of synchronization amongst the cell population When the kinetics for the phosphorylation of IjBa by IKK were measured, we found that the values for the affinity and catalytic efficiency of IKK2 for IjBa were dependent on assay conditions The application of these kinetic parameters in our computational model of the NF-jB pathway resulted in significant differences in the oscillatory patterns of NF-jB depending on the rate constant value used Hence, interpretation of in silico models should be made in the context of this uncertainty In silico analysis of complex cellular processes (whether for data description, drug discovery, genetic engineering or scientific discovery) with its focus on elucidating system mechanisms, has become critical for progress in biology [1–5] Detailed computational models can reveal complex behavior [6] in signaling pathways [7– 9] For example, under certain conditions, signaling molecules can undergo periodic translocation between different cellular compartments resulting in sustained oscillations of their local concentrations [10–12] This has been demonstrated for the nuclear transcription factor nuclear factor (NF-jB), whose nuclear concentration has been shown to oscillate due to translocation to ⁄ from the cytoplasm For the oscillations to be observable in a cell population rather than a single cell, they need to be largely synchronous [13–15] Of course, with the more recent availability of experimental capabilities to inspect single cells dynamically [16], more and more cells have been seen to exhibit asynchronous oscillations [11,12,17] Intact cells like yeast cells can synchronize their oscillations with each other [14], and theoretical studies have demonstrated synchronization (of e.g metabolic pathways) in communicating cells [15] Experimental observations of oscillations have also been made for the p53 [18,19] and mitogen-activated protein kinase [9] signaling pathways, and can also be seen in mathematical models of such processes Abbreviations IKK, IkappaB kinase; IL-1a, interleukin-1a; MeOH ⁄ EtOH ⁄ PEG, methanol ⁄ ethanol ⁄ polyethylene glycol; NF-jB, nuclear factor kappa B; SC-514, 4-amino-2,3¢-bithiophene-5-carboxamide 1678 FEBS Journal 274 (2007) 1678–1690 ª 2007 Pfizer Global Research & Development Journal compilation ª 2007 FEBS A E C Ihekwaba et al [7,9,18,20,21] It is clear that the complexity of biological systems and the difficulty (or at least infrequency) of obtaining kinetic parameters require the development of new analytical methods for both in vitro and in silico biology [22] A still bigger challenge is the measurement of in vivo values of kinetic constants, which may differ critically from their in vitro biochemical counterparts Oscillations have been demonstrated in a variety of components of the NF-jB signaling pathway in single cells [11,12] These results agreed with in silico simulations of the downstream region of the pathway that was modeled, as well as suggesting that the oscillatory frequency has functional significance for downstream events, such that the signal is not simply encoded in its amplitude [23] Activation of the transcription factor NF-jB can be triggered by exposure of cells to a multitude of external stimuli, including the cytokines tumor necrosis factor (TNF-a) and interleukin-1a (IL-1a), thus initiating numerous and diverse intracellular signaling cascades, most of which activate the IkappaB kinase (IKK) complex This crucial component in the NF-jB activation cascade typically consists of two catalytic subunits [24,25], IKKa (IKK1) and IKKb (IKK2) [26–29] and a regulatory unit NF-jB essential modulator (NEMO, IKKc) [30–33] The cytoplasmic inhibitors of NF-jB (the IjBs [34,35]) are phosphorylated by activated IKK at specific N-terminal residues, tagging them for polyubiquitination and rapid proteasomal degradation This allows NF-jB to be released upon activation and it translocates to the nucleus where it induces the transcription of a large number of target genes encoding regulators of immune and inflammatory responses and also genes involved in apoptosis and cell proliferation [36] In this paper, we report the results of cell-based, in vitro and in silico experiments on the NF-jB pathway First, we demonstrate oscillations in a population of 10 000 A549 cells, which is consistent with synchronous behavior Secondly, we present in vitro kinetic measurements of IKK2 protein kinase We demonstrate that the assay conditions can affect substantially the apparent Km and kcat values of this reaction, whose parameters are known to be important in an existing computational model of the NF-jB pathway Thirdly, we use the aforementioned computational model [10,37] to analyze in silico the effect of the parameter variation discussed above The parameter values chosen for this reaction have a significant effect on the amplitude (but not the frequency) of the oscillations Finally, we extend this in silico and in vitro strategy to a cell-based approach to analyze the effect of a known inhibitor of IKK2, 4-amino-2,3¢-bithiophene-5-carbox- In vitro analysis of NF-jB signaling pathway amide (SC-514) [38] We initially performed an in vitro study which confirmed the competitive nature of the inhibition and the published IC50 value Surprisingly, we then found that cells pretreated with inhibitor displayed oscillations of a similar strength (frequency) to those observed in the untreated cell population In order to shed light on the cause of this result, we carried out an in silico analysis by incorporating the inhibition kinetics within the existing computational model This showed that the SC-514 inhibitor has limited impact on the dynamics of NF-jB activation Results and Discussion Immunocytochemistry Immunocytochemical staining of cell-based NF-jB proteins was used to study oscillatory patterns in NF-jB nuclear ⁄ cytoplasmic localization in A549 cells In a previous study, an EC50 of 0.340 ngỈmL)1 for IL-1a was established (Fig 1A,B; data not published); in this case, EC50 is a measure of the IL-1a concentration required to produce 50% of maximal response To investigate if different fixatives and types of the culture substrate have an effect on the intensity of nuclear NF-jB observed, a population time lapse study of nuclear NF-jB translocation following cell stimulation with ngỈmL)1 IL-1a was examined Firstly, we compared the use of a 96-well plastic-bottomed plate with methanol ⁄ ethanol ⁄ polyethylene glycol (MeOH ⁄ EtOH ⁄ PEG) fixative (with 12 repeats for each time point to minimize error as a result of background noise) and glass-bottomed plates with 3.7% formaldehyde fixative affected the quality of the stained images Using immunocytochemical analysis, significant differences in the peak intensity was observed between the two assays The comparison at the 30-min time point revealed peak intensity of nuclear NF-jB to be 2.94 arbitrary units for the MeOH ⁄ EtOH ⁄ PEG fixative and plastic culture plate combination (Fig 1G) and 5.75 arbitrary units for the formaldehyde fixative and glass culture plate combination (Fig 1F) These results indicated that the use of a combination of formaldehyde fixative and glass culture plates produces a better resolved image (higher signal ⁄ noise ratio), giving a better dynamic range of output values when compared with the use of the alcohol-based fixative and plastic culture plates We recently showed asynchronous oscillation following cell stimulation across four single cells [12,39] It has been previously suggested that population-based analyses may not always reveal oscillatory behavior that is occurring on the single-cell level, because pro- FEBS Journal 274 (2007) 1678–1690 ª 2007 Pfizer Global Research & Development Journal compilation ª 2007 FEBS 1679 In vitro analysis of NF-jB signaling pathway A E C Ihekwaba et al B A Translocation Ratio Translocation Ratio (Arbitary units) 500 400 300 200 EC50 =0.34ng·mL–1 100 0.001 0.01 0.1 10 IL-1alpha conc (ng·mL–1) EC50 = 0.148ng·mL–1 0.001 100 0.01 C D 5.9 100 E 3.1 glas s / form aldehyde 5.7 2.9 F 5.5 plastic / MeOH/EtOH/PEG G 2.7 5.3 2.5 [N F -κ B ]n [NF-κ B ]n 0.1 10 IL-1alpha conc (ng·mL–1) 5.1 2.3 4.9 4.7 2.1 4.5 1.9 4.3 1.7 4.1 1.5 50 100 150 200 250 300 350 400 450 Time (min) 50 100 150 200 250 300 350 400 450 Time (min) Fig Immunocytochemical staining of A549 cells and analysis of the dynamics of NF-jB nuclear translocation (A,B) Dose–response data from A549 stimulated with IL-1a and fixed with formaldehyde (A) and MeOH ⁄ EtOH ⁄ PEG (B) (data unpublished) (C–E) Cell images showing nuclear cytoplasmic localization of NF-jB in stimulated (D) and stimulated after pretreatment with SC-514 inhibitor following 30 of IL-1a exposure Cytoplasmic localization of NF-jB in nonstimulated cells is shown in (C), whereas in (D), localization of NF-jB is primarily in the nucleus Arrows draw attention to the localization of NF-jB In (E), NF-jB is observed in both the nuclei and the cytoplasm of the cells (F,G) Time course plot of A549 cells stimulation with ngỈmL)1 IL-1a generated on glass-bottomed plates with 3.7% formaldehyde fixative (F) and clear-bottomed plastic plates with MeOH ⁄ EtOH ⁄ PEG fixative (G) The peaks are the fluorescent intensity of nuclear NF-jB when compared with cytoplasmic NF-jB The error bars in (A), (B), (F) and (G) display standard deviations tein extracts average potentially asynchronous responses of individual cells [40] Despite this, we observed discernible oscillations in the overall levels of NF-jB activation in a population of 10 000 A549 cells suggesting a significant degree of synchronicity (Fig 1F,G) Note that the immunocytochemical approach used does not facilitate the tracking of individual cells over time [40] Having previously shown by sensitivity analysis [37,41] that the oscillatory behavior of nuclear NF-jB is tightly coupled to just two participating species, free 1680 IKK2 and free IjBa, and that reactions such as the phosphorylation of IjBa by IKK exerted a major controlling influence on the amplitude [42] of the oscillations in the computational model [12,37], we next studied the rate of IjBa phosphorylation by IKK Enzyme kinetics of rhIKK2 for glutathioneS-transferase-IjBa We investigated the kinetics of rhIKK2 Figure 2A shows a typical progress curve for the IKK catalyzed FEBS Journal 274 (2007) 1678–1690 ª 2007 Pfizer Global Research & Development Journal compilation ª 2007 FEBS A E C Ihekwaba et al In vitro analysis of NF-jB signaling pathway 0.028 0.026 0.024 0.022 0.02 0 18 0.016 0 14 0.012 0.01 0.008 0.006 0.004 0.002 0.2 (nmoles) [γ-33P]-ATP bound 0.4 (nmoles) [γ-33P]-ATP bound A 0 20 40 60 80 100 B 120 20 40 60 Time (min) 100 C 60 40 D 60 40 0 20 40 60 22 10 12 14 16 12 14 16 [ GST - IκBα ] ( µM) 20 E 20 [ATP] (µM) F 18 16 14 12 10 16 14 nM min-1 Initial velocity (v) 18 nM min-1 120 20 20 Initial velocity (v) 100 80 nM min-1 Initial velocity (v) 80 nM min-1 Initial velocity (v) 100 80 Time (min) 12 10 6 4 2 20 40 60 10 [ GST - IκBα ] ( µM) [ATP] (µM) Fig Enzyme kinetics of rhIKK2 for substrate ATP and GST-IjBa (A) Interaction between rhIKK2 and GST-IjBa (rhIKK2 in vitro kinase assay coupled with GST-IjBa as described in Experimental procedures); Vmax is 1.11 · 10)3 lMỈmin)1, Ks is · 10)3 ± 1.4 · 10)3 lM (B) The control rhIKK2 in vitro kinase assay with no GST-IjBa substrate The time (in minutes) on the abscissa indicates the time the reactions were stopped with trichloroacetic acid and the plot shows the number of repeats Phosphorylation of tagged IjBa (d; A) and autophosphorylated rhIKK2 (j; B) is shown In (A), s is the control assay, and in (B), s and D represent the control repeats, and j represents the average of the two Kinase activities were recorded as incorporation of c-33P (countsỈmin)1) into GST-IjBa (A) and IKK2 (B) (C, D) Michaelis–Menten plots generated by varying [ATP][60 lM (s), 30 lM (d), 15 lM (h), 7.5 lM (j), 3.75 lM (D), 1.88 lM (m), 0.94 lM (Ñ), 0.47 (.)] at fixed [GST-IjBa] (C), and varying [GST-IjBa] [15.33 lM (s), 7.67 lM (d), 3.83 lM (h), 1.92 lM (j), 0.96 lM (D), 0.48 lM (m), 0.24 lM (Ñ), 0.12 lM (.) at a fixed [ATP] (D) Reactions (45 lL, plate assay) were performed at room temperature for 70 with [c-33P]ATP (2.4 lCi).(E, F) Enzyme kinetics of recombinant IKK2 for substrate ATP and GST-IjBa with MnCl2 in Tris ⁄ HCl ⁄ MgCl2 kinase buffer Michaelis–Menten plots generated by varying [ATP] at 15.33 lM GST-IjBa (E), and varying [GST-IjBa] at 60 lM ATP (F) Km,ATP, Km,GST-IjBa, kcat and Vmax were 2.3 ± 0.6 lM, 3.7 ± 0.9 lM, 1.51 · 10)3 s)1 and 18.7 nMỈmin)1, respectively, in kinase buffer Tris ⁄ HCl ⁄ MgCl2 ⁄ MnCl2 and 2.5 ± 1.2 lM, 6.1 ± 1.3 lM, 2.15 · 10)3 s)1 and 16.2 nMỈmin)1 in kinase buffer Hepes ⁄ MgCl2 ⁄ MnCl2 (data not shown) phophorylation of GST-IjBa Figure 2B shows the corresponding control without GST-IjBa These data are consistent with limited autophosphorylation Figure 2C shows plots of rhIKK2 velocity as a function of varying concentration of ATP (0.47–60 lm) at eight fixed concentrations of GST-IjBa (0.12–15.33 lm) We found rhIKK2 displayed standard Michaelis-Menten kinetics at each GST-IjBa concentration with an apparent Km,ATP value of 9.6 ± 3.5 lm (Fig 2C and Table 1) We further examined the kinase activity of FEBS Journal 274 (2007) 1678–1690 ª 2007 Pfizer Global Research & Development Journal compilation ª 2007 FEBS 1681 In vitro analysis of NF-jB signaling pathway A E C Ihekwaba et al Table Michaelis–Menten kinetics, maximal turnover rates for rhIKK2, the limiting maximal velocity and the ratio of apparent dissociation constants for binding GST-IjBa in the presence and app absence of ATP K m is the apparent dissociation constant for full length GST-IjBa substrate at saturation concentration of 60 lM ATP, and the dissociation constant for ATP at saturation concentraapp tion of 15.33 lM GST-IjBa The apparent Vmax (V max) at 60 lM ATP and 50 nM IKK2 is 136 ± 4.2 nMỈmin)1 kcat (s)1) · 10)2 GST-IjBa ATP app Km (lM) app V max (nMỈmin)1) a 1.13 ± 0.016 1.13 ± 0.016 3.8 ± 1.7 9.6 ± 3.5 136 ± 4.2 136 ± 4.2 0.9 ± 0.5 0.9 ± 0.5 rhIKK2 as a function of varying concentrations of GST-IjBa (0.12–15.33 lm) at eight fixed concentrations of ATP (0.47–60 lm) Analyses showed the apparent Km,GST-IjBa, value to be 3.8 ± 1.7 lm (Fig 2D and Table 1), at saturated ATP concentration (six-fold of Km,ATP) We also found the apparent maximal turnover rates (kcat) for rhIKK2 to be 1.13 · 10)2 s)1 at room temperature under the same conditions Previously determined kinetics for IKK2 (Table 2), revealed a 10–52-fold and 30–140-fold variation in the Km values estimated for IjBa and ATP, respectively The wide variation in these reported Km values may be attributed to the use of rhIKK2, nonrhIKK2 or IKK complex, and also different experimental conditions The Km that we determined for GSTIjBa is comparable to a number of previously published values within this wide range [28,43–47] Similarly, our result for Km,ATP is in agreement with some of the values reported in the literature [43,47,48] A noteworthy difference in the previously reported experiments is the presence [33,38,45,46,49–51] or absence [43,44,47,48] of MnCl2 in the assay conditions (i.e MgCl2 with MnCl2 vs MgCl2 only) We therefore decided to perform a second investigation of the Km values in the presence of MnCl2, but with all other experimental conditions constant Comparison with the values already obtained in the absence of MnCl2 would therefore enable us to quantify this effect on two key parameters (as determined by us [37]) in our in silico model Kinetic analysis showed Km,ATP, Km,GST-IjBa, kcat and Vmax to have values of 2.3 ± 0.6 lm, 3.7 ± 0.9 lm, 1.51 · 10)3 s)1 and 18.7 nmỈmin)1, respectively, using Tris ⁄ HCl ⁄ MgCl2 ⁄ MnCl2 buffer and 2.5 ± 1.2 lm, 6.1 ± 1.3 lm, 2.15 · 10)3 s)1 and 16.2 nmỈmin)1 in the kinase assay using Hepes ⁄ MgCl2 ⁄ MnCl2 buffer These findings confirmed the importance of kinase conditions used for determining kinetic values (Fig 2E,F) A list of previously established kinetic values for IKK2 is reviewed in Table Having shown that the disparity in the experimental kinetic results is dependent on the kinase condition used, we next studied how the experimental kinetic data reported here affected the NF-jB model previously described [12,37,39] Substitution of the rates with the kinetic values reported in this section and Table showed a more damped oscillatory pattern, similar to (see Fig 3H in [52]) and with comparable frequency to the original model (see Fig 3D) These findings indicate that substituting previously reported kinetic data in the original model with the experimental data determined here results in an oscillatory pattern analogous to that seen in the population time study of the A549 cells (Fig 1F,G, where the Table A list of kinetic constants for IjBa and ATP substrates with IKK2 rh, recombinant human IKK2; nonrh, nonrecombinant human IKK2; norm, IKK complex; Y, present Km (ATP) (lM) Km (IjBa) (lM) 7.3 0.13 0.05 1.3 2.2 2.6 1.4 1.7 0.5 2.2 0.94 0.7 3.83 6.1 3.7 15.5 0.13 0.56 18 0.65 0.6 9.6 2.5 2.3 1682 kcat (s)1) · 10)3 4.5 21.0 37.0 0.92 3.5 4.56 11.2 11.3 2.15 1.51 Type of IKK2 Buffer MgCl2 nonrh norm rh rh norm rh norm rh rh rh rh rh rh Tris ⁄ HCl Hepes Tris ⁄ HCl Tris ⁄ HCl Hepes Tris ⁄ HCl Hepes Hepes Hepes Hepes Tris ⁄ HCl Hepes Tris ⁄ HCl Y Y Y Y Y Y Y Y Y Y Y Y Y MnCl2 Y Y Y Y Y Reference [48] [45] [28] [43] [46] [44] [33] [47] [49] [51] Y Y FEBS Journal 274 (2007) 1678–1690 ª 2007 Pfizer Global Research & Development Journal compilation ª 2007 FEBS A E C Ihekwaba et al A B 100 % CONTROL 80 (nmoles) [γ-33P]-ATP bound In vitro analysis of NF-jB signaling pathway 60 40 20 0 10 100 1000 1 10 [INHIBITOR] C 1000 6.1 untreated A549 stimulated cells 5.6 [NF- κ B]n 100 [INHIBITOR] pre-treated A549 stimulated cells 5.1 4.6 4.1 3.6 50 100 150 200 250 300 350 400 450 Time (min) D E 0 Ori ginal model 0 O r ig inal model New model Inhibitor model 0.08 New model 0 0.07 0 [NF-κ B]n [N F - κ B ]n 0.1 0.09 0 0 0.06 0.05 0.04 0 0.03 0 0.02 0 0.01 0 50 100 150 250 200 Time (min) 300 350 400 450 50 100 150 200 250 300 350 400 450 Time (min) Fig Effect of SC-514 inhibitor on the activity of rhIKK2 homodimer (A,B) Different concentrations of SC-514 inhibitor was incubated with recombinant IKK2, and an IC50 experiment was undertaken using 10 lM (A, h), lM (B, d) and 0.1 lM (B, s) ATP as described in Experimental procedures (C) Time lapse of nuclear cytoplasmic localization of NF-jB in 10 000 A549 cells These cells were dispensed onto a Whatman 96 glass bottomed plates and treated with ngỈmL)1 IL-1a in the presence and absence of the SC-514 inhibitor (D, E) Time course plot of nuclear NF-jB from in vitro and in silico analysis of the data The plot shows nuclear NF-jB oscillation in the original model and in the updated model with newly measured kr1, ka1 and kd1 for IKKIjBa complex (D) In the original model kr1 (kcat) for IKKIjBa was 4.07 · 10)3 s)1 In the updated model, the original values are replaced with 1.13 · 10)2 s)1 (D) (E) shows a plot of the original and the updated model with the inclusion of newly measured Ki in the updated model amplitudes are damped) We have thus far demonstrated that kinase assay conditions affect the experimental rate values We have also substantiated that the oscillatory pattern of the model is affected when the new data is implemented in the model We next studied the impact of a rhIKK2 inhibitor on the oscillatory pattern of both cell-based and in silico nuclear NF-jB translocation Effect of SC-514 Inhibitor on cell-based nuclear NF-jB translocation Kishore et al [38] first characterized the selective inhibitor SC-514 in 2003, and showed that it inhibited all forms of recombinant human IKK2 including rhIKK2 homodimer and rhIKK1 ⁄ IKK2 complex FEBS Journal 274 (2007) 1678–1690 ª 2007 Pfizer Global Research & Development Journal compilation ª 2007 FEBS 1683 In vitro analysis of NF-jB signaling pathway [38,53,54] A comparable IC50 value for rhIKK2 was obtained in the present study to that previously reported by Kishore et al [38] We obtained values of 0.13 ± 0.06 lm for 0.1 lm ATP, 0.17 ± 0.08 lm for lm ATP and 5.61 ± 0.65 lm for 10 lm ATP (Fig 3A,B) This shift in IC50 values confirms the competitive nature of this SC-514 inhibitor It was also observed that a concentration of 100 lm of SC-514 is sufficient to completely inhibit IjBa degradation by IKK2 in vitro (Fig 3A,B); this was also as established by Kishore et al [38] Having shown SC-514 to inhibit IjBa phosphorylation, thus demonstrating an inhibition of IKK2 activity in vitro, we next determined whether SC-514 would inhibit activated native IKK complex in IL-1a-stimulated A549 cells To test whether these in vitro data were also found in in vitro cell cultures, a nuclear NF-jB translocation assay was performed where the cells were pretreated with 100 lm of the SC-514 inhibitor We examined the effect of SC-514 treatment on NF-jB activation by stimulating A549 cells with IL-1a for 400 In the presence of SC-514, the kinetics of NF-jB activation and inactivation with the IL-1a was observed Immunofluorescence analysis showed that following a 6-h exposure with IL-1a, NF-jB translocated from the cytoplasm to the nucleus in the entire A549 population, irrespective of their pretreatment with SC-514 inhibitor (Fig 3C) Figure 3C displays time-course plots of nuclear NF-jB dynamics for pretreated and untreated A549-stimulated cells Interestingly, the pretreated cells displayed clearly discernible oscillations that closely followed those of the untreated cells in terms of their frequency The effects of exposing the cells to the inhibitor were slight, amounting to a modest reduction in amplitude and a delay in the first oscillatory peak It is interesting to speculate on the failure of the inhibitor to eliminate the oscillations or at least substantially dampen them A similar apparent discrepancy between in vitro and cell-based results was also reported by Kishore et al [38], who reported some phosphorylation of IjBa even after SC-514 pretreatment at a level (100 lm) that caused complete in vitro inhibition One possible explanation is that this inhibitor does not block the activity of another IKK isoform, IKK1 Consequently, IjBa may still be phosphorylated by the IKK1 isoform when the IKK1 isoform is present and activated in the system Another could be that the intracellular concentrations of ATP (Mg) are high enough to attenuate observed inhibition Alternatively, it may well be the case that IKK is not the only point of regulation in the NF-jB pathway [55], and that IjBa phosphorylation and degradation, and the subsequent translocation of NF-jB into the nucleus may be mediated by 1684 A E C Ihekwaba et al another mechanism Such a mechanism could be derived from the theory underlying metabolic control analysis, which states that the control exerted by individual parameters depends not only on their own magnitude but also on that of all the others [56,57] Effect of SC-514 Inhibitor on in silico nuclear NF-jB translocation To investigate whether the observed in vitro and cellbased inhibition data translated to an in silico effect, we examined the impact of the determined experimental inhibition kinetic data on the same model previously described by Ihekwaba et al [37] Inclusion of our experimentally determined rate constant (Ki 0.114 lm; kcat 11.3 · 10)3 s)1) resulted in a dampened oscillatory pattern with a frequency similar to that of the original model (Fig 3E) Interestingly, the inclusion of IKK2 inhibition by SC-514 with our experimentally determined rate constants in the original model resulted in a delay in simulated peak and damping of subsequent peaks (Fig 3E), a feature also observed in the cell-based assay (Fig 3C) One implication of this finding is that the effects of future inhibitors designed for this NF-jB signaling pathway should be tested not just in vitro and cell based but also simulated in silico Combining this method of analysis (in vitro, cell-based and in silico analysis) will facilitate systematic understanding of the underlying properties of this signaling pathway To summarize Activation of cells via stimuli, TNF-a [12] and IL-1a [58] induces activation of the NF-jB transcription factor The consequences of how changes in external stimuli influenced a cascade of co-operative events were assessed in vitro, in cell cultures, and also in silico in this study Previous work [12] demonstrated oscillatory behavior in the levels of nuclear NF-jB in single cell studies In our cell-based experiments, a population of 10 000 A549 cells was observed to undergo similar oscillatory behavior to that discovered in single cells in terms of the peak periods and frequency This clearly demonstrated that these cells have the ability to synchronize their oscillations with each other The study of signaling pathway dynamics requires detailed cell-based measurements of time-varying phenomena, in this case, oscillatory variations of nuclear levels of NF-jB In order to attain this degree of precision, optimization of experimental conditions and techniques is required In the past three decades, FEBS Journal 274 (2007) 1678–1690 ª 2007 Pfizer Global Research & Development Journal compilation ª 2007 FEBS A E C Ihekwaba et al advances in cell culture techniques and immunocytochemistry have enabled the explanation of diverse immunological phenomena We evaluated two alternative immunocytochemical experimental protocols for cell assay analysis and found one to be superior for the scope of this study Better resolved immunocytochemical images were obtained using glass bottomed plates with formaldehyde fixative rather than plasticbottomed plates with MeOH ⁄ EtOH ⁄ PEG fixative A quantitative assay to measure the phosphorylating activity of rhIKK2 enzymes with GST-IjBa substrate was described It was observed that subtle changes in the experimental design had a profound effect on the kinetic data obtained Kinase assay environment was shown in this study to have a significant effect on the Km,GST-IjBa, Km,ATP, and the Vmax found, and cautions us to take appropriate measures when choosing rate values from the literature The importance of choosing the relevant kinetic parameters when building a computational model was also demonstrated in this study We have shown that the inhibition of IKK2 blocks response in vitro Despite the fact that IKK2 has been identified as a key participant in the NF-jB signaling pathways, both our cell-based and in silico studies revealed that this inhibition has limited impact on the dynamics of NF-jB activation It should be stressed that the in silico model presented here represents a considerable simplification of the NF-jB signaling pathway For example, it does not consider participants upstream of IKK2, or other putative mechanisms for regulation of nuclear NF-jB Nevertheless, the findings presented in this paper demonstrate that even a simplified computational model can give us a deeper understanding of the complex system behavior of such signaling pathways The key findings indicate that computational modeling can be a useful complement to biochemical and imaging experiments The results reported in this paper should encourage further synergistic experimental and computational studies aimed towards elucidating other complex signaling systems Experimental procedures Materials Materials and apparatus, and their suppliers, were as follows: formaldehyde (3.7%) in NaCl ⁄ Pi (internal stores, Pfizer Global Research and Development, Sandwich, UK); MeOH ⁄ EtOH ⁄ PEG [60% v ⁄ v 95% EtOH, 20% v ⁄ v MeOH (HPLC quality), 7% v ⁄ v PEG (Sigma Aldrich, Gillingham, UK); NaCl ⁄ Pi, pH 7.2 (Invitrogen, Paisley, UK)]; polyoxyethylene sorbitan monolaurate (Sigma); Draq nuclear In vitro analysis of NF-jB signaling pathway stain (Biostatus Ltd., Shepshed, UK); Cellomics NF-jB Hit kit Evaluation ⁄ Screening (Cellomics Inc., Pittsburg, PA, USA); DMEM (Gibco, Invitrogen); 200 mm l-glutamine; fetal bovine serum (Gibco Invitrogen, Virkon, Pfizer internal stores) IL-1a (R & D Systems, Minneapolis, MN, USA); Whatman 96-well sterile tissue culture treated glass bottomed plates; 96-well, clear-bottomed plastic plates (Costar); Gilson p200 yellow, p1000 blue and p10 pink tips (Gilson, Middleton, WI, USA); Reagent boats and Falcon tubes Recombinant IKK2 was donated by Frank Stuhmeier of the Hit Discovery Group (HDG laboratory at Pfizer) Other reagents and apparatus used were as follows: GSTIjBa fusion protein [c-33P]ATP (Amersham Bioscience, Chalfont St Giles, UK); ATP (Roche diagnostics GmbH, Mannheim, Germany); trichloroacetic acid; 50 mm Tris ⁄ HCl pH 7.5, 10 mm MgCl2; 50 mm Hepes pH 7.5, 10 mm MnCl2; NaCl ⁄ Pi (Invitrogen); Microscint 40 (Packard, Waltham, MA, USA); Plate seals (Packard); 96-well white microplate with bonded GF ⁄ C filter [unifilter 96, GF ⁄ C (Perkin Elmer)]; microtiter plate (Millipore Corp.) All other reagents and apparatus were of high quality available from Sigma sources Cell culture A549 cells (human lung carcinoma epithelial cell line SNB0000178-CE A549) were passaged every days in DMEM (+ mm l-glutamine and 5% fetal bovine serum) and maintained at 37 °C and 5% CO2 For translocation experiments, cells were removed with 0.05% trypsin ⁄ EDTA, and plated with cell solution of · 106 cellsỈmL)1 (in a 50 mL flask) and grown until 80% confluency Cellular assays A549 cell solution (100 lL) was seeded on a plastic, flat-bottomed · 96-well- plates (Coster) at a density of · 104 cells per well and incubated for an 18–24-h period at 37 °C and 5% CO2 A solution of IL-1a (concentration 40 ngỈmL)1 resulting in a final concentration of ngỈmL)1 per well due to the : dilution factor) was prepared for the time-course assay Stimulation of cells was performed at 10-min intervals for 400 with IL-1a After 400 min, the plates were inverted to remove media into a dish containing Virkon disinfectant to destroy cells not adhered to the plates MeOH ⁄ EtOH ⁄ PEG fixation solution (100 lL; prewarmed in a water bath at 37 °C) was dispensed into each well and incubated for 15 (prewarming fixative is critical to maintaining cell integrity) After 15 min, the plates were inverted to remove the fixation solution, and 100 lL of NaCl ⁄ Pi was dispensed into the wells The plates were next inverted to remove the NaCl ⁄ Pi wash solution, 100 lL of permeabilization buffer was then dispensed into the wells and left to incubate for 90 s at room temperature The plates were again inverted to remove the permeabilization buffer, FEBS Journal 274 (2007) 1678–1690 ª 2007 Pfizer Global Research & Development Journal compilation ª 2007 FEBS 1685 In vitro analysis of NF-jB signaling pathway and washed twice with NaCl ⁄ Pi thereby removing wash buffer by inverting the plates Rabbit polyclonal immunoglobulin IgG (50 lL; primary antibody) was dispensed into each well and left to incubate for h at room temperature The plates were inverted to remove antibody after the h incubation period, 100 lL of detergent [NaCl ⁄ Pi and 0.1% Tween 20 (polyoxyethylene sorbitan monolaurate)] was dispensed into the wells and the plates left to incubate for 15 The plates were inverted to remove the detergent after the 15 incubation period and the wells were washed twice with wash buffer by inverting the plates Staining solution (50 lL; containing goat antirabbit IgG conjugated to Alexa Fluor 488 secondary antibody and Draq5 dye; or and Hoechst 33258 dye) was dispensed into each well and left to incubate for h at room temperature in the dark The plates were inverted to remove the antibody solution and 100 lL of detergent dispensed into the wells and left to incubate for 10 The plates were inverted to remove detergent solution and 100 lL of wash solution dispensed into the wells The plates were inverted to remove the wash solution for the last time and replaced with 200 lL of wash buffer The plates were sealed and analyzed on Evotec OPERA (Evotec, Hamburg, Germany) This assay study was also repeated with a glass flat-bottomed · 96-well plates (Whatman) and 4% formaldehyde fixative Immunocytochemical analysis On reading a microplate using the NF-jB protocol, the Evotec OPERA has been programmed to find the nuclei centers of the cells by using the DRAQ5 or Hoechst 33258 nuclear stain image DRAQ5 is excited with 633 nm laser and its peak emission is 685 nm, whilst Hoechst uses nearUV excitation (380 nm) and gives blue emission (530 nm) The software was used according to the manufacturer’s instructions (Scheme 1) A E C Ihekwaba et al containing 100 lgỈmL)1 ampicillin A colony of E coli in LB agar plates was inoculated into 50 mL of LB liquid medium and incubated on shaking platform with 200 r.p.m at 37 °C for h The value at D600 measured by spectrophotometry was used to indicate the bacterial concentrations Inoculated liquid medium (2 · 25 mL) was added into a · 500 mL of LB liquid medium, and incubated on rotator with 200 r.p.m at 37 °C for 1.5 h The value at D600 was again measured by spectrophotometry The glutathione-Stransferase fusion proteins were induced by · 500 lL of mm isopropyl b-d-1-thiogalactopyranoside addition to the E coli medium and finally incubated on a rotator with 200 r.p.m at 37 °C for h The bacterial cells in the · 500 mL medium were harvested by centrifugation (27 500 r.p.m for 10 min, °C, Beckman rotor) Collected bacteria were re-suspended in a · 25 mL NaCl ⁄ Pi buffer The re-suspended cell mixture was placed in a disrupter machine with NaCl ⁄ Pi and 2-mercaptoethanol (total collected volume ẳ 120 mL) Benzoase (125 unitsặmL)1) added to the collected viscous liquid The collected liquid was centrifuged, the separated soluble fusion protein filtered (volume collected ¼ 110 mL) and purified using immobilized metal chromatography at mLỈmin)1 (absorbance of collected liquid using IMAC ¼ A280) The supernatant was loaded onto a glutathione affinity column according to the manufacturer’s protocol Bound glutathione-S-transferase proteins eluted with mm glutathione in NaCl ⁄ Pi (and 2-mercaptoethanol) GST-IjBa (6 mL) eluted from the column Protein concentrations measured in a Bradford (Bio-Rad, Hercules, CA, USA) protein assay Peak fraction were pooled and subjected to 12% Tris-glycine SDS ⁄ PAGE and western analysis to determine the purity of the GST-IjBa Glycerol (2 mL) was added to prevent damage from freezing, and the end volume was transferred into Eppendorf tubes in aliquots of 400 lL Kinase time-course assay Cloning, expression and purification of GST-IjBa fusion proteins To overexpress the protein GST-IjBa, the plasmid vectors were transformed into BL21 (DE3) Escherichia coli strains, and the cells were grown overnight in 10 mL LB medium Intensity of cytoplasm Ratio of Translocation = Intensity of nucleus Intensity of cytoplasm Intensity of nucleus Scheme This is a simplification of a cell as seen by analysis software, where the software measures the intensity of NF-jB in the nucleus when compared with the intensity of NF-jB in the cell 1686 Recombinant human IKK2 (rhIKK2) time-course reaction was carried out for 113 in 50 mm Tris ⁄ HCl, pH 7.5, and 10 mm MgCl2 Reactions were performed in a final volume of 45 lL (15 lL of rhIKK2, 15 lL ATP [c-33P]ATP, 15 lL GST-IjBa for kinase assay and 15 lL of rhIKK2, 15 lL ATP [c-33P]ATP, 15 lL 50 mm Tris ⁄ HCl pH 7.5, 10 mm MgCl2 for control assay) For experiments related to Ks determination of rhKK2 and GST-IjBa binding, assays were carried out with 50 nm IKK2, lm GST-IjBa peptide, 0.05 lCi [c-33P] ATP (10 mCiỈmmol)1) and 0.2 lm ATP Reaction mixture was withdrawn and dispensed into a 96-well white microplate with bonded GF ⁄ C filter [unifilter 96, GF ⁄ C (Perkin Elmer)] Each well was successively washed five times with 100 lL of 12% w ⁄ v trichloroacetic acid, once with 100 lL lm ATP, twice again with 100 lL 12% w ⁄ v trichloracetic acid, and once with 100 lL 50 mm Tris ⁄ HCl, pH 7.5, and 10 mm MgCl2 The plate was FEBS Journal 274 (2007) 1678–1690 ª 2007 Pfizer Global Research & Development Journal compilation ª 2007 FEBS A E C Ihekwaba et al allowed to dry for 10 in a 55 °C oven, and then 35 lL of scintillation fluid (Microscint 40) was dispensed to each well Incorporated [c-33P]ATP was measured using a Top count NXT (Packard) The amount of IKK-catalyzed incorporation of 33P into each peptide was quantified by liquid scintillation counting The counts represent initial velocity of rhIKK2-catalysed phosphorylation (< 30% of total ATP conversion) The graphs were fitted using grafitTM software, and k1 and k2 were calculated from Vmax and Ks values expressed in unitsỈmol)1 of enzyme per and unitsỈmol)1, respectively Kinase assay with Tris ⁄ HCl ⁄ MgCl2 rhIKK2 kinase reactions were carried out for 70 in 50 mm Tris ⁄ HCl, pH 7.5, and 10 mm MgCl2 The amounts of substrates ATP, [c-33P]ATP (10 mCiỈmmol)1; Amersham Bioscience), and GST-IjBa are specified for each individual experiment Reactions were performed in a final volume of 45 lL (15 lL of rhIKK2, 15 lL ATP, [c33 P]ATP, 15 lL À GST-IjBa) For experiments related to Km determinations of IKK2, various concentrations of ATP and GST-IjBa peptide were used in the assay at a fixed concentration of either GST-IjBa or ATP For GST-IjBa peptide Km, assays were carried out with 50 nm IKK2, 60 lm ATP, 2.4 lCi [c-33P]ATP (10 mCiỈmmol)1) and GST-IjBa peptide from 0.12 to 15.33 lm For ATP Km, assays were carried out with 50 nm IKK2, 15.33 lm GST-IjBa peptide, lCi [c-33P]ATP (10 mCiỈmmol)1) and ATP from 0.47 to 60 lm Sample was analyzed by precipitation on a microtiter plate (Millipore Corp) For the microtiter plate assays, 45 lL of reaction sample ⁄ well was precipitated with 45 lL of 12% w ⁄ v trichloroacetic acid 70 lL of the reaction mixture was withdrawn and dispensed into a 96-well white microplate with bonded GF ⁄ C filter (unifilter 96, GF ⁄ C; Perkin Elmer) Washing of precipitated sample was performed using the same protocol as that described for the kinase time-course assay The assay was again repeated with the inclusion of 10 mm MnCl2 in the kinase condition In vitro analysis of NF-jB signaling pathway Tris ⁄ HCl, pH 7.5, and 10 mm MgCl2 and typically included: 50 ng of rhIKK2; varying concentrations of SC-514 inhibitor [300–0.1 lm; reconstituted 2.688 mg of SC-514 (relative molecular mass 224 g) to mL of 12 000 lm stock solution in 100% dimethyl sulfoxide]; and 5.11 lm GSTIjBa peptide per well at 10 lm ATP lCi [c33 P]ATP À (10 mCiỈmmol)1), lm ATP 0.1 lCi [c33 P]ATP (10 À mCiỈmmol)1) and 0.1 lm ATP 0.05 lCi [c33 P]ATP À (10 mCiỈmmol)1) separate ATP concentrations, to make a total volume of 40 lL (rhIKK2 10 lL, SC-514 10 lL, ATP 10 lL and GST-IjBa 10 lL) The reaction was run in duplicate A positive and a negative control assay were also included, where the positive control contains no inhibitor in the assay and the negative control was stopped at time zero Reaction sample (40 lLỈwell)1) was precipitated with 40 lL of 12% w ⁄ v trichloroacetic acid Reactions were performed using the same protocol as that described for the Tris ⁄ HCl ⁄ MgCl2 and Hepes ⁄ MgCl2 ⁄ MnCl2 assay Kinetic analysis For two substrate profile analysis, initial velocity studies were performed with varying concentrations of GST-IjBa at several fixed concentrations of ATP and vice versa (order of binding experiments) Lineweaver–Burk double reciprocal plots were generated by linear least squares fits of the data Replotting the slopes and the y intercepts of the lines as function of ⁄ [ATP] generated secondary plots Kinetic constants (Km for ATP, GST-IjBa, and Vmax) values were determined from a global fit to the database using erithacus software grafit 4- where Vmax is the limiting maximal velocity that would be observed when all the enzyme is present as enzyme–substrate ‘ES’ [rhIKK2-GST-IjBa], Km is the Michaelis–Menten constant and the kcat is the breakdown of the ES complex to E + product (P) [59] (Eqn 1) The equilibria describing competitive inhibition of the SC-514 are show in Eqn 2, where Ki is the dissociation constant for the enzyme–inhibitor (EI) complex To obtain 50% (IC50) inhibition, refer to Eqn [59] k1 Kinase assay with Hepes ⁄ MgCl2 ⁄ MnCl2 rhIKK kinase reactions were carried out for 70 in 50 mm Hepes pH 7.5, and 10 mm MgCl2 and 10 mm MnCl2 The amounts of substrates, ATP, [c-33P]ATP (10 mCiỈmmol)1, Amersham Bioscience) and GST-IjBa were the same as those specified in the assay with Tris ⁄ HCl ⁄ MgCl2 Reactions were performed using the same protocol as that described for the Tris ⁄ HCl ⁄ MgCl2 assay IC50 ([I]0.5) dose–response assay IC50 experiments were performed in 96-well Millipore plates The reactions were carried out for 45 in 50 mm kcat E ỵ S ! ES E ỵ P ! ỵ k2 I #" Ki EI Ki ẳ IC50 =ẵ1 ỵ ẵS=Km ފ ½1Š ½2Š For a random sequential model, values for Km,ATP, Km,GST-IjBa, Vmax and a was determined from the global fit The constant a is the ratio of apparent dissociation constants for binding GST-IjBa in the presence and absence of ATP, and the value of a indicates whether the binding of one substrate (ATP) affects the affinity of the enzyme for the other substrate (GST-IjBa) [59] FEBS Journal 274 (2007) 1678–1690 ª 2007 Pfizer Global Research & Development Journal compilation ª 2007 FEBS 1687 In vitro analysis of NF-jB signaling pathway SC-514 inhibition cellular assays A549 cells were pretreated with 100 lm SC-514 inhibitor IKK2 inhibitor (25 lL, 600 lm; SC-514) (100 lmỈwell)1 due to the : dilution factor) was dispensed into the wells of each plate prior to IL-1a addition A total of 48 ngỈmL)1 (8 ngỈmL)1 per well due to the : dilution factor for wells containing IKK2 inhibitor) of IL-1a was prepared for cell assay and was added following the same procedure described for the time course cell assay The cells were washed with NaCl ⁄ Pi and fixed after 400 min, and the standard immunocytochemical detection applied The plates were sealed and analyzed using an Evotec OPERA confocal micro plate imaging reader (Evotec) Mathematical modeling All simulations were performed using gepasi and copasi simulators, initially with parameters described in the revised supplemental information for Hoffmann et al [10] (http:// www.sciencemag.org/cgi/data/298/55965596/1241/DC1/2); including the pre-equilibration period of 2000 s A diagram of the network can be obtained from [12,37,41] Parameters were varied using the ‘scan’ function in gepasi The mathematical model described here has been submitted to the online Cellular Systems Modeling Database and can be accessed at http://jjj.biochem.sun.ac.za/database/ihekwaba/ index.html free of charge Acknowledgements We thank everyone at Discovery Biology (HDG) Pfizer (PGRD) Sandwich, especially, Simon Eaglestone for providing the GST-IjBa, Frank Stuheimer for providing the recombinant IKK2 (rhIKK2) and Nandini Kishore for providing the SC-514 inhibitor We thank also Paul Hayter, Sasha Sreckovic and Matthew Strawbridge with help in growing and analyzing the A549 cells and finally BBSRC for the award of a CASE studentship to AECI References Chakraborty AK, Dustin ML & Shaw AS (2003) In silico models for cellular and molecular immunology: successes, promises and challenges Nat Immunol 4, 933–936 Westerhoff HV & Palsson BO (2004) The evolution of molecular biology into systems biology Nat Biotechnol 22, 1249–1252 Tyson JJ, Chen KC & Novak B (2003) Sniffers, buzzers, toggles and blinkers: dynamics of regulatory and signaling pathways in the cell Curr Opin Cell Biol 15, 221–231 Butcher EC, Berg EL & Kunkel EJ (2004) Systems biology in drug discovery Nat Biotechnol 22, 1253–1259 1688 A E C Ihekwaba et al Moore MN & Noble D (2004) Editorial: computational modelling of cell 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components of the NF-jB signaling pathway in single cells [11,12] These results agreed with in silico simulations of the downstream region of the pathway that was modeled, as well as suggesting that the